lymphoma 2; Bim, Bcl2-interacting mediator
of cell death) ( 6 ). Bim was encoded into the
latch as a sequestered peptide, and Bcl2 was
used as the effector. We added targeting do-
mains that recruit the Co-LOCKR cage and
key to cells expressing target antigens. While
the targeting domains should bind to any cell
expressing their target antigens, only cells with
both antigens should colocalize cage and key
(Fig. 1C). Because Co-LOCKR is thermodynam-
ically controlled, complex formation occurs at
much lower solution concentrations when
the components are bound to and colocalized
on a surface than when they are unbound (fig.
S3, A and B); colocalization shifts the bind-
ing equilibrium in favor of complex forma-
tion (fig. S3C).
To evaluate the ability of Co-LOCKR to tar-
get cells coexpressing a precise combination
of surface antigens, we developed a mixed-
population flow cytometry assay by combining
four K562 cell lines expressing Her2-eGFP,
EGFR-iRFP, both, or neither (eGFP, enhanced
green fluorescent protein; EGFR, epidermal
growth factor receptor; iRFP, near-infrared
fluorescent protein) (Fig. 1D). We used de-
signed ankyrin repeat protein (DARPin) do-
mains ( 7 , 8 ) to target the cage and key to Her2
and EGFR, respectively. If the system func-
tions as designed, only cells coexpressing both
Her2 and EGFR should activate Co-LOCKR
and bind Bcl2 (Fig. 1E): The cage contains
the sequestered Bim peptide, and the key is
required for its exposure. We refer to this Co-
LOCKR configuration as CL_CHKE; in this
nomenclature, CL refers to Co-LOCKR, CH
indicates that the cage is targeted to Her2,
and KEindicates that the key is targeted to
EGFR (table S2). When the mixed population
of cells was coincubated with an equimolar
dilution series of cage and key (3mM to 1.4 nM)
and washed before adding AlexaFluor594-
labeled Bcl2 (Bcl2-AF594), the expected sig-
moidal binding curve was observed for the
Her2-EGFR cells but not for cells expressing
either antigen alone (Fig. 1, F and G).
We next sought to tune the dynamic range of
Co-LOCKR activation to increase colocalization-
dependent activation sensitivity and respon-
siveness. The sensitivity of previous LOCKR
switches was tuned by shortening the latch to
produce a“toehold,”which allows the key to
outcompete the latch ( 3 ), but this also pro-
moted aggregation (fig. S2, bottom). We there-
fore focused on designing mutations rather
than toeholds to tune the relative interaction
affinities of the Co-LOCKR system to be co-
localization dependent (fig. S4). We mutated
large, hydrophobic residues in the latch [Ile^287 →
Ala (I287A), I287S, I269S] or cage (L209A) to
weaken cage–latch affinity (Fig. 2A). Biolayer
interferometry indicated that increasingly dis-
ruptive mutations improved responsiveness
(fig. S5, A and B), and flow cytometry showed1640 25 SEPTEMBER 2020•VOL 369 ISSUE 6511 sciencemag.org SCIENCE
Fig. 3. Co-LOCKR performs two- and three-input logic operations in mixed cell populations.
(A) Co-LOCKR was used to recruit Bcl2-AF594 in mixed populations of K562 cells expressing different
combinations of Her2, EGFR, and EpCAM. Marker expression for each cell line and identity of the cage
and key targeting domains are indicated below each bar plot. Red highlighting indicates the expected
magnitude of Bcl2-AF594 signal based on relative antigen expression. (B) Schematic of [Her2 AND either
EGFR OR EpCAM] logic mechanism. (C) [Ag 1 AND either Ag 2 OR Ag 3 ] logic combinations were used to
recruit Bcl2-AF594. (D) Schematic of [Her2 AND EpCAM NOT EGFR] logic mechanism. The decoy
acts as a sponge to sequester the key, thereby preventing cage activation. (E) CL_CHKEpDEwas used to
recruit Bcl2-AF594. Compared with the simple CL_CHKEpAND gate (left), recruitment of decoy to
EGFR-expressing cells reduced activation to near background levels. For allpanels, population 1 was
[K562-EpCAMlo, K562-EGFR-EpCAMlo, K562-Her2-EpCAMlo, and K562-Her2-EGFR-EpCAMlo], and
population 2 was [K562-EpCAMlo, K562-EGFR-EpCAMlo, K562-Her2-EpCAMhi, and K562-Her2-EGFR-
EpCAMhi]. Fluorescence intensities are normalized to the negative control. Error bars represent SEM of
six independent replicates for K562 and K562-EGFR and three independent replicates for all others.
Statistics are reported in table S4.
RESEARCH | REPORTS